The present invention relates generally to quantitative cell mutagenesis, and more particularly to a fluorometric method of quantitative cell mutagenesis.
Quantitative mutagenesis in a cell culture can be determined by staining the culture with a colorimetric or histochemical stain, followed by observance of the adsorption curve obtained utilizing a measuring instrument such as a spectrophotometer. For example, if the mutagenesis is caused by the absence of a specific enzyme in cells, the cell culture is first stained colorimetrically. The normal cells will exhibit absorbance at a particular wavelength, while the cells deficient in the enzyme will not be absorbing at this wavelength.
A colorimetric method of cell mutagenesis detection is described by A. Wajntal and R. DeMars in Biochemical Genetics 1:61-64 (1967). Specifically, a histochemical method for the detection of the enzyme glucose-6-phosphate dehydrogenase (G6PD) is described. Enzymatic reduction of NADP to NADPH in the presence of the substrate (glucose-6-phosphate) is coupled to the reduction of tetrazolium salts. Colored, insoluble deposits appear within cells containing the enzyme. A similar staining technique is described in Proc. Nat. Acad. Sci. USA, Vol. 72, No. 2, pp 493-497 (1975). The production of NADPH, and hence the presence of the G6PD, is determined by observing the increase in absorbence at 340 mm on a recording spectrometer. The assaying of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT), utilizing a colorimetric staining method, is described by L. A. Chasin in Cell, 2, 37-41 (1974).
Colorimetric staining assay methods as described above include a number of serious disadvantages. Mutagenesis, e.g., cells deficient in a cellular compound, can be expected to occur typically once in a culture of 100,000 cells. With the methods described above, the absorbance at a particular wavelength of each cell is observed. Detection of the abnormal cell is dependent on the observance of a difference, such as a pronounced decrease in staining intensity, thereby pinpointing the mutagenetic cell. Noise signals inherent in all detection instruments tend to interfere in the detection. Thus the reliability of the results is suspect. Additionally, the colorimetric methods require plating and growing of clones which may take 10 days or more to achieve.
Instead of detecting a drop in colorimetric activity at an occurrence level of one out of every 100,000 cells, it is more desirable to detect only those cells where mutagenesis has occurred. Staining, followed by fluorescent counter-staining of cell cultures, provides such a detection method. This method permits cell assaying to be completed in approximately three hours, and minimizes problems associated with instrument background noise.